Takato Terada

Trehalose-Enhanced Fluidity of the Goat Sperm Membrane and Its Protection During Freezing

Abstract In an attempt to find a suitable freezing method for goat semen, two experiments were conducted to study the influence of trehalose on the cryopreservation of goat spermatozoa. In experiment 1, goat spermatozoa were frozen in trehalose extender (0.375 M) alone (100%) or at different combinations of trehalose with Tris-citric acid-glucose (TCG) extender (0%, 25%, 50%, 75%). Final concentrations of 20% (v:v) egg yolk and 4% (v:v) glycerol were employed in the extenders (osmolality = 370, pH = 7). Sperm motility was assessed using a computer-assisted sperm analysis system and acrosome integrity was assessed using fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA). The sperm-motility parameters improved significantly by increasing the concentration of trehalose (P < 0.05) and significantly high recovery rates for the motility parameters were also achieved by a high concentration of trehalose (P < 0.05). Motility of the frozen-thawed spermatozoa after a 3-h incubation improved significantly with increasing concentrations of trehalose in the extender (P < 0.05). The 75% and 100% trehalose extenders yielded a significant increase in the percentage of spermatozoa with intact acrosome (P < 0.05). In experiment 2, merocyanine 540/Yo-Pro staining was used to study the influence of trehalose on membrane fluidity compared with that of sucrose and TCG. Percentage of cells with high merocyanine fluorescence was significantly higher in spermatozoa treated with trehalose than sucrose or TCG (P < 0.05), indicating a significantly highest membrane fluidity of sperm samples extended with trehalose solution. We thus conclude that the substitution of a Tris-citric acid diluent composition with trehalose significantly improves the freezability of goat spermatozoa. Furthermore, the cryoprotective effects of trehalose observed in this study may be due to enhanced sperm membrane fluidity before freezing.

Production of Progesterone from De Novo-Synthesized Cholesterol in Cumulus Cells and Its Physiological Role During Meiotic Resumption of Porcine Oocytes

Abstract To investigate the role of factors secreted by cumulus cells during meiotic resumption of porcine oocytes, 1, 5, 10, or 20 cumulus-oocyte complexes (COCs) were cultured in each well of a culture dish containing 300 μl of maturation medium for 20 h. There was a significant positive correlation between the rate of germinal vesicle breakdown (GVBD) and the number of COCs cultured in each well for 20 h. The level of progesterone in the medium in which COCs had been cultured for 20 h also rose significantly with an increase in the number of COCs cultured in each well. A significantly small proportion of GVBD in oocytes when one COC was cultured in each well for 20 h was improved by the addition of progesterone. This proportion of GVBD was fully comparable to that of COCs cultured in the absence of additional progesterone with 20 COCs. Thus, progesterone secreted by COCs plays a positive role in GVBD induction in porcine oocytes. Furthermore, we also examined the role of sterol biosynthesis on progesterone production by cumulus cells and in oocyte GVBD. The results showed that the addition of ketoconazole, which suppressed the sterol biosynthetic pathway produced by demethylation of lanosterol, decreased the rate of GVBD, as well as progesterone production in COCs cultured for 20 h. However, the suppression of GVBD by ketoconazole was overtaken by the addition of progesterone. These results demonstrate that a high level of progesterone produced by cumulus cells was responsible for an acceleration of GVBD in porcine oocytes.

Luteinizing Hormone Receptor Formation in Cumulus Cells Surrounding Porcine Oocytes and Its Role During Meiotic Maturation of Porcine Oocytes

Abstract We investigated the formation of LH receptor (LHR) in cumulus cells surrounding porcine oocytes and the role of LHR in meiotic maturation of oocytes. At least three splice variants of LHR mRNA were detected in cumulus cells, in addition to the full-length form. Low levels of three types of products were seen in cumulus cells from cumulus oocytes complexes (COCs), whereas the full-length form was significantly increased by 12-h cultivation with FSH. The addition of FSH also significantly increased the binding level of biotinylated hCG to COCs. The formation of LHR in FSH-stimulated cumulus cells was not affected by additional 0.5 mM phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and the oocytes were synchronized to the germinal vesicle (GV) II stage by exposure to 0.5 mM IBMX and FSH for 20 h. The binding of LH to its receptor induced a further increase in cAMP level and progesterone production and acceleration of meiotic progression to the metaphase I stage. The oocytes cultured with LH for 24 h following cultivation with FSH and IBMX were used for in vitro fertilization. At 6 days after in vitro fertilization, blastocyst rate in oocytes matured under these conditions was significantly higher than that of oocytes cultured in the absence of LH. Treatment of oocytes with FSH and 0.5 mM IBMX to express LH receptor in cumulus cells while holding oocytes at the GV II stage is a very beneficial way to produce in vitro-matured oocytes, which have high developmental competence.

Dynamic Changes of Connexin-43, Gap Junctional Protein, in Outer Layers of Cumulus Cells Are Regulated by PKC and PI 3-Kinase During Meiotic Resumption in Porcine Oocytes

Abstract Mammalian oocytes are surrounded by numerous layers of cumulus cells, and the loss of gap junctional communication in the outer layers of cumulus cells induces meiotic resumption in oocytes. In this study, we investigated the dynamic changes in the gap junctional protein connexin-43 in cumulus cells during the meiotic resumption of porcine oocytes. The amount of connexin-43 in all layers of cumulus cells recovered from cumulus-oocyte complexes was increased after 4-h cultivation. However, at 12-h cultivation, the positive signal for connexin-43 immunoreactivity was markedly reduced in the outer layers of cumulus cells. When these reductions of connexin-43 were blocked by protein kinase C (PKC) or phosphatidylinositol (PI) 3-kinase inhibitor, networks of filamentous bivalents (i.e., advanced chromosomal status) were undetectable in the germinal vesicle of the oocyte. After 28-h cultivation, when the majority of oocytes were reaching the metaphase I (MI) stage, the connexin-43 in the inner layers of cumulus cells was phosphorylated, regardless of mitogen-activated protein (MAP) kinase activation. These results suggest that the initiation of meiotic resumption, namely, the formation of networks of filamentous bivalents in germinal vesicle, is associated with the reduction of gap junctional protein connexin-43 in the outer layers of cumulus cells via the PKC and/or PI 3-kinase pathway. Moreover, the connexin-43 in the inner layers of cumulus cells is phosphorylated during meiotic progression beyond the MI stage, regardless of MAP kinase activation in cumulus cells surrounding the oocyte.

Phosphatidylinositol 3-Kinase in Cumulus Cells and Oocytes Is Responsible for Activation of Oocyte Mitogen-Activated Protein Kinase During Meiotic Progression Beyond the Meiosis I Stage in Pigs

Abstract The roles of phosphatidylinositol 3-kinase (PI 3-kinase) during meiotic progression beyond the meiosis I (MI) stage in porcine oocytes were investigated. PI 3-kinase exists in cumulus cells and oocytes, and the PI 3-kinase inhibitor, LY294002, suppressed the activation of mitogen-activated protein (MAP) kinase in denuded oocytes during the beginning of the treatment. However, in denuded oocytes cultured with LY294002, the MAP kinase activity steadily increased, and at 48 h of cultivation MAP kinase activity, p34cdc2 kinase activity, and proportion of oocytes that had reached the meiosis II (MII) stage were at a similar level to those of oocytes cultured without LY294002. In contrast, LY294002 almost completely inhibited the activation of MAP kinase, p34cdc2 kinase activity, and meiotic progression to the MII stage in oocytes surrounded with cumulus cells throughout the treatment. Treating cumulus oocyte complexes (COCs) with LY294002 produced a significant decrease in the phosphorylation of connexin-43, a gap junctional protein, in cumulus cells compared with that in COCs cultured without LY294002. These results indicate that PI 3-kinase activity in cumulus cells contributes to the activation of MAP kinase and p34cdc2 kinase, and to meiotic progression beyond the MI stage. Moreover, gap junctional communications between cumulus cells and oocytes may be closed by phosphorylation of connexin-43 through PI 3-kinase activation in cumulus cells, leading to the activation of MAP kinase in porcine oocytes.

Effect of Protein Kinase C Activator on Mitogen-Activated Protein Kinase and p34cdc2 Kinase Activity During Parthenogenetic Activation of Porcine Oocytes by Calcium Ionophore

Abstract The objective of this study was to elucidate the role of a [Ca2+]i rise and protein kinase C (PKC) activation on decreases of p34cdc2 kinase and mitogen-activated protein (MAP) kinase activity during parthenogenetic activation of porcine oocytes. In oocytes treated with 50 µM Ca2+ ionophore, degradations of both p34cdc2 kinase and MAP kinase activity were observed and half of these oocytes formed pronuclei. However, a supplement of PKC inhibitor, calphostin C, after 50 µM Ca2+ ionophore treatment, was sufficient to inhibit the inactivation of MAP kinase and pronuclear formation in the oocytes. These results showed that PKC played an important role in Ca2+-induced oocyte activation. On the other hand, 10 µM Ca2+ ionophore treatment could not affect the MAP kinase activity but induced a transient decrease of p34cdc2 kinase activity, which resulted in recovery of p34cdc2 kinase activity and progression to meiotic metaphase III stage. To investigate the effects of PKC activator on oocytes treated with 10 µM Ca2+ ionophore, matured oocytes were cultured with phorbol 12-myriatate 13-acetate (PMA), after 10 µM Ca2+ ionophore treatment. The additional treatment suppressed the recovery of p34cdc2 kinase activity and rapidly induced a decrease of MAP kinase activity, and these low activities were maintained until 12-h cultivation. As a result, a significantly higher percentage of these oocytes (67%) had pronuclei at 12-h cultivation. Moreover, PMA treatment without Ca2+ ionophore treatment effectively led to a decrease of MAP kinase activity in a dose-dependent manner but not p34cdc2 kinase activity in matured porcine oocytes. In conclusion, the parthenogenetic activation of porcine oocytes was mediated by the inactivation of p34cdc2 kinase via a calcium-dependent pathway and thereafter by the inactivation of MAP kinase via a PKC-dependent pathway.

Higher rates of development into blastocyst following the in vitro fertilization of bovine oocytes matured in a medium supplemented with the fluid from large bovine follicles

Abstract The effects of increasing amounts (0–60%) of bovine follicular fluid (bFF) from large follicles ( ≥ 15 mm in diameter) added to medium supplemented with 10% (v/v) oestrous cow serum (MSES) (TCM 199+LH+FSH+10% oestrous cow serum) on in vitro maturation of bovine follicular oocytes and on their subsequent development to the blastocyst and hatched blastocyst stages were investigated. After 24 h of incubation, the oocytes were fertilized and then cultured for 10 days. The effects of addition of oestrous cow serum (ECS) to maturation medium (at 15%) on development of the oocytes were also examined. Bovine follicular fluid had no stimulatory effect on the rate of nuclear maturation. However, supplementation of maturation medium with 10 or 15% bFF resulted in significantly higher proportions of morulae, blastocysts and hatched blastocysts. Although supplementation of maturation medium with ECS was also beneficial for development of bovine oocytes, probably, the interactions between the factors in bFF and ECS prevented achievement of an additive effect when both fluids were added at this concentration. In conclusion, the results suggest that the fluid from large bovine follicles contains stimulatory substance (s) that during in vitro maturation promotes the potential of bovine follicular oocytes for the subsequent development to blastocysts and hatched blastocysts.

Studies of the role of steroid hormone in the regulation of oocyte maturation in cattle.

BackgroundThe objective of this study was to investigate whether the steroid hormone(s) secreted from cumulus-oocyte complexes (COCs) is a prerequisite for bovine oocyte maturation and cumulus expansion using aminoglutethimide (AGT), a P450 cholesterol side-chain cleavage inhibitor.MethodsIn experiment 1, COCs were cultured in maturation medium with various concentrations of AGT for 22 h to determine the effective concentration of AGT to inhibit steroid hormone secretion, meiotic maturation and cumulus expansion. In experiment 2, COCs were cultured in conditioned medium (CM) and TCM-199 medium with or without 10 mM AGT to check whether steroid hormones secreted from COCs were responsible for oocyte maturation and cumulus expansion. Experiments 3 and 4 were carried out to determine whether exogenous progesterone or estradiol-17beta was able to overcome the inhibitory effects of AGT on oocytes maturation and cumulus expansion. COCs cultured in 10 mM AGT-containing medium supplemented with various concentrations of progesterone or estradiol-17beta for 22 h were examined for oocyte maturation and cumulus expansion.ResultsExperiment 1 showed that a concentration of 10 mM AGT in medium was sufficient to block steroid hormone secretion, oocyte maturation and cumulus expansion, and that these inhibitory effects were fully reversible. In experiment 2, the addition of 10 mM AGT to CM did not significantly prevent oocyte maturation and cumulus expansion, implying that CM contains the steroid hormone(s) secreted from COCs, which are closely associated with oocyte maturation and cumulus expansion. The results in experiments 3 and 4 demonstrated that the addition of any concentration of progesterone or estradiol-17beta in the medium did not reduce the inhibitory effects of AGT on oocyte maturation and cumulus expansion.ConclusionOur results indicate that bovine oocytes surrounded by cumulus cells are prevented from maturation and cumulus expansion through the inhibition of steroid secretion due to AGT, and that these inhibitory effects of AGT on oocyte maturation and cumulus expansions can not be overcome by the addition of either progesterone or estradiol-17beta in the medium. These observations suggest that some steroid hormone(s) other than P4 and E2 secreted from bovine COCs is essential for their meiotic maturation and cumulus expansion.

Survival of boar spermatozoa frozen in diluents of varying osmolality

We investigated the effects of freezing diluents of differing levels of osmolality on boar sperm cryosurvival. The spermatozoa were frozen using a pellet technique. Cryosurvival was evaluated in terms of motility, intact acrosomes and membrane integrity. The motility parameters were assessed using a computer-assisted sperm motility analysis (CASA) system. Acrosomal status was monitored by means of FITC-labeled peanut agglutinin, and membrane integrity was evaluated after double staining with SYBR-14 and propidium iodide. At 3 h of incubation after thawing, the highest motility was found in the 420 mOsm/kg group, and progressive motiLity in the 420 to 580 mOsm/kg groups was higher than that in the hypo- (225 mOsm/kg) and iso-osmotic (290 mOsm/kg) groups (P < 0.05). The intact acrosomes of the spermatozoa frozen in the 510 and 580 mOsm/kg BF5 diluents were more numerous than in other groups (P < 0.05). The 420 and 510 mOsm/kg groups yielded maximal values of post-thaw membrane integrity. These observations obtained in the present study indicate that moderately hypertonic BF5 diluents are favorable for the cryopreservation of boar spermatozoa.

Mitogen-Activated Protein Kinase Kinase Inhibitor Suppresses Cyclin B1 Synthesis and Reactivation of p34cdc2 Kinase, Which Improves Pronuclear Formation Rate in Matured Porcine Oocytes Activated by Ca2+ Ionophore

Abstract To investigate the role of mitogen-activated protein (MAP) kinase kinase (MEK)/MAP kinase cascade on p34cdc2 kinase activity and cyclin B1 levels during parthenogenetic activation of porcine oocytes, MEK activity, MAP kinase activity, p34cdc2 kinase activity, and cyclin B1 levels were assayed in mature porcine oocytes after treatment with different concentrations of Ca2+ ionophore. A high concentration of Ca2+ ionophore (50 μM) rapidly reduced MEK activity in oocytes for up to 8 h of culture. MEK activity in the 10-μM treatment group was significantly higher. The low concentration treatment transiently decreased p34cdc2 kinase activity but did not affect MAP kinase activity and ultimately induced reactivation of p34cdc2 kinase via the synthesis of cyclin B1. On the other hand, treatments of a high concentration of Ca2+ ionophore or a low concentration of Ca2+ ionophore plus MEK inhibitor, U0126, linearly decreased MAP kinase activity following the decrease of p34cdc2 kinase activity; most of these oocytes formed pronuclei. These results suggest that decreasing MAP kinase activity is essential to maintaining low p34cdc2 kinase activity resulting from the degradation of cyclin B via a Ca2+-dependent pathway; lower activities of both MAP kinase and p34cdc2 kinase induce normal meiotic completion and pronuclear formation of parthenogenetically activated porcine oocytes.